The existence of a peculiar branched RNA-linked DNA molecule called msDNA (multicopy single-stranded) has been demonstrated in various myxobacteria, Gram-negative soil bacteria (Yee et al., 1984; Dhundale et al., 1985; Furuichi et al., 1987; Dhundale et al., 1987; Dhundale et al. 1988 J. Biol. Chem. 48, 47-53 and 55-62. msDNA (msDNA-Mx162) from Myxococcus xanthus consists of 162-base single stranded DNA, the 5' end of which is linked to the 2' position of the 20th rG residue of a 77-base RNA molecule (msdRNA) by a 2', 5'- phosphodiester linkage (Dhundale et al., 1987). It exists at a level of approximately 700 copies per genome. Stigmatella aurantiaca also possesses an msDNA (msDNA-Sa163) which is highly homologous to msDNA-Mx162 (Furuichi et al., 1987). In addition to msDNA-Mx162, M. xanthus has another smaller species of msDNA (mrDNA or msDNA-Mx65), which has no primary sequence homology with msDNA-Mx162 or msDNA-Sa163 (Dhundale et al., 1988 J. Biol. Chem.). However, all msDNAs so far characterized share key structural features such as a branched rG residue, stem-and-loop structures in RNA and DNA molecules, and a DNA-RNA hybrid at the 3' ends of DNA and RNA molecules.
Previously it was predicted that reverse transcriptase is required for msDNA biosynthesis on the basis of the finding that msdRNA is derived from a much longer precursor, which can form a very stable stem-and-loop structure (Dhundale et al., 1987). This precursor molecule was proposed to serve as a primer for initiating msDNA synthesis as well as a template to form the branched RNA-linked-msDNA. The latter reaction requires reverse transcriptase activity. In M. xanthus, the region coding for the RNA molecule (msr) is located on the chromosome in the opposite orientation to the msDNA coding region (msd) with the 3' ends overlapping by 6 bases for msDNA-Mx65 (Dhundale et al., 1988 J. Biol. Chem.) or by 8 bases for msDNA-Mx162 (Dhundale et al., 1987). In addition, as in all the msDNAs found in myxobacteria, there is an inverted repeat comprised of a 14-base sequence for msDNA-Mx65 (Dhundale et al., 1988 J. Biol. Chem.) or a 34-base sequence for msDNA-Mx162 (Dhundale et al., 1987) and a 33-base sequence for msDNA-Sa163 (Furuichi et al., 1987) immediately upstream of the branched G residue and a sequence immediately upstream of the msDNA coding region. As a result of this inverted repeat, a longer primary transcript beginning upstream of the RNA coding region and extending through the msDNA coding region is considered to self-anneal and form a stable secondary structure. When three base mismatches were introduced into the secondary structure immediately upstream of the branched rG residue, msDNA synthesis was almost completely blocked. However, if three additional base substitutions were made on the other strand to resume the complementary base pairing, msDNA production was restored (Hsu et al., 1989). This result strongly supports the proposed model for msDNA synthesis.
It has also been shown that a deletion mutation at the region 100 base pairs (bp) upstream of the DNA coding region (msd) and an insertion mutation at a site 500 bp upstream of msd caused a significant reduction in msDNA production (Dhundale et al., 1988 J. Bacteriol.). This indicates that there is a cis- or trans-acting positive element required for msDNA synthesis in this region. In this report we determined the DNA sequence of this region and found an open reading frame (ORF) of 485 amino acid residues beginning with an initiation codon, ATG, which is located 77 bp upstream of msd (or 231 bp downstream of msr). The very close proximity between msd and the ORF suggests that they may be transcribed as a single transcript. The amino acid sequence of the ORF shows similarity with retroviral reverse transcriptases. A possible origin of the reverse transcriptase gene as well as a possible relationship between the msDNA system and retroviruses is discussed herein. Recently, some strains of Escherichia coli were found to produce msDNA and the gene for reverse transcriptase which is essential for msDNA production, is linked to the msd region, (Lim and Maas, 1989; Lampson et al., 1989 Science). Comparison of the msDNA systems of M. xanthus and E. coli raises an intriguing question as to how the extensive diversity found in msDNA systems has emerged in bacteria and what possible functions msDNA may have.
It is shown in U.S. Pat. No. 5,079,151 that msDNA is in fact synthesized by reverse transcriptase in a cell-free system in M. xanthus (Lampson et al., 1989 Cell).